The goal of the Program Project, of which this is a component, is to improve transplantation by decreasing the cell surface expression of major histocompatibility complex (MHC) class I and II proteins. These proteins are the primary antigenic stimuli that trigger the host immunologic rejection response. The approach taken is to treat the graft tissue or organ ex vivo, pretransplant, with oligonucleotides which block the expression of MHC proteins. Oligonucleotides that act by mechanisms including antisense duplex formation, antigene triplex formation, aptamer protein binding, or decoy binding of proteins required for transcription are used. The goal of this specific project is to improve transplantation through covalent modifications of oligonucleotides to increase their access to intracellular targets. Most of the oligonucleotide internalized by most cells is contained within endoplasmic vesicles, and thus cannot reach its cytoplasmic and/or nuclear target. Two strategies will be followed in parallel to increase cytoplasmic delivery of oligonucleotides, from which they can access both cytoplasmic and nuclear targets. The first strategy will modify the backbone phosphates of an oligonucleotide to cause it to partition into membrane lipid and diffuse through the membrane directly into the cytoplasm, and thereby mimic a native macromolecular transport system. The feasibility of this novel strategy will be tested using H-phosphate chemistry and phosphoramidate oligonucleotides. Oligonucleotides will be initially screened for their ability to bind to cells and be partially sequestered from the external media as determined by KI quenching of fluorescently labeled oligonucleotides. If feasibility is demonstrated, prodrug constructs will be synthesized in which the delivery-enhancing modifying groups are removed by intracellular enzymes to generate unmodified oligonucleotide within the cell. The second strategy will tether a small molecule (pyrene, polyethylene glycol, or a polyamine) to an oligonucleotide that is anticipated to cause increased internalization by an endocytotic route. This is a traditional strategy that apparently relies on the oligonucleotide that leaks from endocytotic vesicles to reach intracellular targets. The small molecules can also increase the apparent potency of an oligonucleotide by stabilizing the oligonucleotide target complex and by increasing the nuclease resistance of the oligonucleotide. Delivery of oligonucleotide into the cytoplasm and nucleus of HeLa cells will assessed using Texas Red-labeled fluorescent oligonucleotides and confocal microscopy. The ability of modified oligonucleotides to better block MHC protein expression and diminish the host immune response will be evaluated in model systems.